1. Technical Field
The present invention is directed to an in vitro aqueous composition comprising a drug, preferably tacrolimus, having enhanced stability. The invention utilizes a binding protein to stabilize the drug in an aqueous matrix.
2. Background
Many drugs which are administered for therapeutic purposes require an accurate and precise analysis of the measurement of the drug in a bodily fluid. Typically, such analytical methods utilize a composition of the drug in one or more calibrators or control solutions. These solutions are used, for example, to create a calibration curve against which the patient sample (having an unknown concentration of the drug of interest) can be accurately extrapolated. In addition, the quality control schemes of most assay systems are predicated on using stable drug standards to assess assay reliability.
As is often the case the drug standard is supplied as one component of an assay kit and thus it is desirable that the kit components are stable to the rigors of transportation, handling and storage. For the ease of use of the technician, it is preferred to provide the assay standards as an essentially aqueous composition. However, many of these same drugs are not stable in aqueous environments and thus tend to degrade rapidly in aqueous matrices. Exemplary among such drugs which are not stable in aqueous environments are the immunosuppressant drugs tacrolimus (also known as FK506) and rapamycin. The development of commercial diagnostic assays are hampered by such unstable compounds. Overcoming these problems represent a challenge in the development of diagnostic assays.
Both tacrolimus and rapamycin have been used as immunosuppressants to control tissue rejection following transplant therapy. Thus, similar to that which is done during cyclosporine therapy, the monitoring of blood concentration of these drugs may be an important aspect of clinical care.
EP 0 293 892 describes an ELISA methodology to measure FK506 (tacrolimus) comprised of 1 ) an ELISA plate coated with anti-FK506 antibodies, 2) an FK506-horseradish peroxidase conjugate which competes with free FK506 and acts as a signal generating reagent and 3) an appropriate substrate for the peroxidase. The protocol requires that the control solution of FK506 used in the assay is made just prior to use from an aliquot of FK506 stored in ethanol at 2.degree.-8.degree. C.
U.S. Pat. No. 5,338,684to Grenier, et al describes a stabilized composition of FK506 wherein the drug is stored in the presence of whole or lysed blood. The patent discloses that such a composition is stable for at least one day at 37.degree. C. when compared to the drug stored in the absence of whole or lysed blood. The composition is said to useful as a standard or calibrator in assays for FK506. However, such a composition suffers from a drawback in the use of blood or blood components in the composition and the resulting potential biohazard created by handling such products.
Binding proteins for drugs in the immunosuppressant class have been reported. Exemplary among the binding proteins for the drugs tacrolimus or rapamycin are U.S. Pat. Nos. 5,196,352, 5,109,112, International Publication Numbers WO 93/07269 and WO 92/18527, and European Patent Publications 0 584 217 and 0482 189.
International Publication Number WO 92/01052 and European Patent Publication 0 481 673 disclose DNA sequences encoding FK506 binding proteins.
International Publication Number WO 93/25533 discloses a recombinant FKBP fusion protein and it's use in an assay for the purification of tacrolimus.
International Publication Number WO 94/04700 discloses a monoclonal antibody which is specific for an antigenic determinant on both FI&lt;506 and FKBP and an assay employing such an antibody.
International Publication Number WO 95/00174 discloses that antibodies may be stabilized during storage by a solution of a protective ligand having low affinity for the antibody.
Accordingly, a need exists for stable in vitro aqueous compositions of drugs which are unstable under aqueous conditions for utilization as, for example, aqueous standards for diagnostic assays for such drugs.